T cell activation in chronic rheumatoid arthritis (RA) is likely to involve antigen-independent mechanisms, as well as antigen-specific responses. Several surface structures, distinct from the CD3-antigen receptor complex, can contribute to, initiate or regulate T cell activation in vitro. The CD6 T lymphocyte glycoprotein is thought to play a role in T cell proliferation, although its exact function and ligand(s) remain unknown. The applicant's laboratories have recently developed a novel anti-CD6 monoclonal antibody, termed UMCD6, using an RA synovial T cell line as the immunogen. UMCD6 can potently activate peripheral blood T cells, and has a broader range of such mitogenic effects than other previously-described anti-CD6 reagents. UMCD6 is one of several tools used by our laboratories to demonstrate dual regulation of CD6 expression, which is increased both by phorbol ester and by inhibitors of DNA-methyltransferase, such as 5- azacytidine. T cell clones exposed to 5-azacytidine become autoreactive, concurrent with selective upregulation of a small number of surface antigens, notably CD6. Moreover, UMCD6 blocks both antigen-specific and autoreactive responses of such clones. Of additional interest, regarding a role for CD6 in T cell activation in RA synovium, is the recent finding that gammadelta, but not alphabeta clones, proliferate in response to anti- CD6. This proposal will therefore address the role of CD6 in both early and chronic RA using several approaches. The ability of anti-CD6 monoclonal antibodies to induce or regulate T cell responses will be examined using autoreactive T cell clones, antigen and superantigen-specific T cell clones of both alphabeta and gammadelta receptor types, and defined T cell subsets from blood and synovial fluid of patients with RA. The ligand for CD6 will be identified, characterized and cloned, using a variety of immunologic and molecular approaches, and its expression and distribution examined in the synovial compartment. Several approaches will be used to isolate a CD6 ligand, which is proposed to exist as a membrane glycoprotein expressed by antigen-presenting cells (APC). CD6 will be immunoaffinity purified, conjugated to biotin, and used as an immunologic probe for the CD6 ligand. Available monoclonal antibodies against appropriate APC will then be screened for ability to block CD6 binding to the putative ligand. Alternatively, new monoclonal antibodies will be generated to identify the ligand, either anti-idiotypic antibodies against anti-CD6, or monoclonal antibodies raised against the relevant APC population. Such antibodies will be used to biochemically characterize the CD6 ligand. The cDNA for a CD6 ligand will then be isolated in a mammalian expression system, using either CD6-biotin or anti-ligand antibody as a screening probe. The proposed studies will provide fundamental information about the role of CD6 in both normal and autoimmune T cell function.